Mycological Progress

, Volume 9, Issue 1, pp 79–100 | Cite as

MALDI-TOF MS of Trichoderma: a model system for the identification of microfungi

  • Sophie De Respinis
  • Guido Vogel
  • Cinzia Benagli
  • Mauro Tonolla
  • Orlando Petrini
  • Gary J. Samuels
Original Article

Abstract

This investigation aimed to assess whether MALDI-TOF MS analysis of the proteome could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of peptide mass fingerprints would reveal apomorphies that could be useful in diagnosing species in this genus. One hundred and twenty nine morphologically and genetically well-characterized strains of Hypocrea and Trichoderma, belonging to 25 species in 8 phylogenetic clades, were analyzed by MALDI-TOF MS mass spectrometry. The resulting peak lists of individual samples were submitted to single-linkage cluster analysis to produce a taxonomic tree and were compared to ITS and tef1 sequences from GenBank. SuperSpectra™ for the 13 most relevant species of Trichoderma were computed. The results confirmed roughly previously defined clades and sections. With the exceptions of T. saturnisporum (Longibrachiatum Clade) and T. harzianum (Harzianum Clade), strains of individual species clustered very closely. T. polysporum clustered distantly from all other groups. The MALDI-TOF MS analysis accurately reflected the phylogenetic classification reported in recent publications, and, in most cases, strains identified by DNA sequence analysis clustered together by MALDI-TOF MS. The resolution of MALDI-TOF MS, as performed here, was roughly equivalent to ITS rDNA. The MALDI-TOF MS technique analyzes peptides and represents a rough equivalent to sequencing, making this method a useful adjunct for determination of species limits. It also allows simple, reliable, and quick species identification, thus representing a valid alternative to gene sequencing for species diagnosis of Trichoderma and other fungal taxa.

Keywords

Taxonomy MALDI-TOF MS Mass spectrometry Hypocreales 

References

  1. Bénitez T, Limón C, Delgado-Jardina J, Rey M (1998) Glucanolytic and other enzymes and their genes. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor & Francis, London, pp 101–128Google Scholar
  2. Bigly P, Tenkanen M (1998) Enzymology of hemicellulose degradation. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor & Francis, London, pp 25–48Google Scholar
  3. Bissett J (1991) A revision of the genus Trichoderma. III. Section Pachybasium. Can J Bot 69:2373–2417Google Scholar
  4. Bright JJ, Claydon MA, Soufian M, Gordon DB (2002) Rapid typing of bacteria using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and pattern recognition software. J Microbiol Meth 48:127–138CrossRefGoogle Scholar
  5. Caballero ML, Gómez M, González-Muñoz M, Reinoso L, Rodríguez-Pérez REA, Moneo I (2007) Occupational sensitization to fungal enzymes used in animal feed industry. Int Arch Allerg Immun 144:231–239CrossRefGoogle Scholar
  6. Chaverri P, Samuels GJ (2003) Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): species with green ascospores. Stud Mycol 48:1–116Google Scholar
  7. Chaverri P, Castelbury LA, Samuels GJ, Geiser DM (2003) Multilocus phylogenetic structure within the Trichoderma harzianum/Hypocrea lixii complex. Mol Phylogenet Evol 27:302–313CrossRefPubMedGoogle Scholar
  8. Chen HY, Chen YC (2005) Characterization of intact Penicillium spores by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 19:3564–3568CrossRefPubMedGoogle Scholar
  9. Crouch JA, Clarke BB, Hillman BI (2006) Unraveling evolutionary relationship among the divergent lineages of Colletotrichum causing anthracnose disease in turfgrass and corn. Phytopathology 96:46–60CrossRefPubMedGoogle Scholar
  10. de Souza JT, Pomella AWV, Bowers JH, Pirovani CP, Loguercio LL, Hebbar KP (2006) Genetic and biological diversity of Trichoderma stromaticum, a mycoparasite of the cacao witches’-broom pathogen. Phytopathology 96:61–67CrossRefPubMedGoogle Scholar
  11. Degenkolb T, Brückner H (2008) Peptaibiomics: towards a myriad of bioactive peptides containing Calpha-dialkylamino acids? Chem Biodivers 5:1817–1843CrossRefPubMedGoogle Scholar
  12. Degenkolb T, Gräfenhan T, Berg A, Nirenberg HI, Gams W, Brückner H (2006a) Peptaibiomics: Screening for polypeptide antibiotics (peptaibiotics) from plant-protective Trichoderma species. Chem Biodivers 3:593–610CrossRefPubMedGoogle Scholar
  13. Degenkolb T, Gräfenhan T, Nirenberg HI, Gams W, Brückner H (2006b) Trichoderma brevicompactum Complex: Rich source of novel and recurrent plant-protective polypeptide antibiotics. J Agric Food Chem 54:7047–7061CrossRefPubMedGoogle Scholar
  14. Degenkolb T, von Döhren H, Nielsen KF, Samuels GJ, Brückner H (2008a) Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin research, and their importance for chemotaxonomy of Trichoderma and Hypocrea. Chem Biodivers 5:671–680CrossRefPubMedGoogle Scholar
  15. Degenkolb T, Gams W, Brückner H (2008b) Natural cyclopeptaibiotics and related cyclic tetrapeptides: structural diversity and future prospects. Chem Biodivers 5:693–706CrossRefPubMedGoogle Scholar
  16. Degenkolb T, Dieckmann R, Nielsen KF, Gräfenhan T, Theis C, Zafari D, Chaverri P, Ismaiel A, Brückner H, von Döhren H, Thrane U, Petrini O, Samuels GJ (2008c) The Trichoderma brevicompactum clade: a separate lineage with new species, new peptaibiotics, and mycotoxins. Mycol Prog 7:177–219CrossRefGoogle Scholar
  17. Dickinson DN, La Duc MT, Satomi M, Winefordner JD, Powell DH, Venkateswaran K (2004) MALDI-TOF MS compared with other polyphasic taxonomy approaches for the identification and classification of Bacillus pumilus spores. J Microbiol Meth 58:1–12CrossRefGoogle Scholar
  18. Domsch KH, Gams W, Anderson T-H (2007) Compendium of soil fungi, 2nd taxonomically revised edition by W. Gams. IHW, EchingGoogle Scholar
  19. Druzhinina IS, Kopchinskiy AG, Komon M, Bissett J, Szakacs G, Kubicek CP (2005) An oligonucleotide barcode for species identification in Trichoderma and Hypocrea. Fungal Genet Biol 42:813–828CrossRefPubMedGoogle Scholar
  20. Druzhinina IS, Kopchinskiy AG, Kubicek CP (2006) The first 100 Trichoderma species characterized by molecular data. Mycosci 47:55–64CrossRefGoogle Scholar
  21. Druzhinina IS, Komon-Zelazowska M, Kredics L, Hatvani L, Antal Z, Belayneh T, Kubicek CP (2008) Alternative reproductive strategies of Hypocrea orientalis and genetically close but clonal Trichoderma longibrachiatum, both capable of causing invasive mycoses of humans. Microbiology 154:3447–3459CrossRefPubMedGoogle Scholar
  22. Evans HC, Holmes KA, Thomas SE (2003) Endophytes and mycoparasites associated with an indigenous forest tree, Theobroma gileri, in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases. Mycol Prog 2:149–160CrossRefGoogle Scholar
  23. Fenselau C, Demirev PA (2001) Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 20:157–171CrossRefPubMedGoogle Scholar
  24. Friedrichs C, Rodloff AC, Chhatwal GS, Schellenberger W, Eschrich K (2007) Rapid identification of Viridans streptococci by mass spectrometric discrimination. J Clin Microbiol 45:2392–2397CrossRefPubMedGoogle Scholar
  25. Fujimori F, Okuda T (1994) Application of the random amplified polymorphic DNA using the polymerase chain reaction for efficient elimination of duplicate strains in microbial screening. I. Fungi. J Antibiot (Tokyo) 47:173–182Google Scholar
  26. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species - opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56CrossRefPubMedGoogle Scholar
  27. Hathout Y, Demirev PA, Ho Y-P, Bundy JL, Ryzhov V, Sapp L, Stutler J, Jackman J, Fenselau C (1999) Identification of Bacillus spores by matrix-assisted laser desorption ionization-mass spectrometry. Appl Environ Microbiol 65:4313–4319PubMedGoogle Scholar
  28. Hermosa MR, Keck E, Chamorro I, Rubio B, Sanz L, Vizcaino JA, Grondona I, Monte E (2004) Genetic diversity shown in Trichoderma biocontrol isolates. Mycol Res 108:897–906CrossRefPubMedGoogle Scholar
  29. Hughes KW, Petersen RH, Mata JL, Psurtseva NV, Kovalenko AE, Morozova OV, Lickey EGB, Blanco JC, Lewis DP, Nagasawa E, Halling RE, Takehashi S, Aime MC, Bau T, Henkel T (2007) Megacollybia (Agaricales). Rep Tottori Mycol Inst 45:1–57Google Scholar
  30. Jaklitsch WM, Samuels GJ, Dodd SJ, Lu B-S, Druzhinina IS (2006) Hypocrea rufa/ Trichoderma viride: a reassessment, and description of five closely related species with and without warted conidia. Stud Mycol 56:135–177CrossRefPubMedGoogle Scholar
  31. Jaklitsch WM, Kubicek CP, Druzhinina IS (2008) Three European species of Hypocrea with reddish brown stromata and gree ascospores. Mycologia 100:796–815CrossRefPubMedGoogle Scholar
  32. Kindermann J, El-Ayouti Y, Samuels GJ, Kubicek CP (1998) Phylogeny of the genus Trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster. Fungal Genet Biol 24:298–309CrossRefPubMedGoogle Scholar
  33. Klein D, Eveleigh DE (1998) Ecology of Trichoderma. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor & Francis, London, pp 57–74Google Scholar
  34. Koivula A, Linder M, Teeri TT (1998) Structure-function relationships in Trichoderma cellulolytic enzymes. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor & Francis, London, pp 3–24Google Scholar
  35. Komon-Zelazowska M, Bissett J, Zafari D, Hatvani L, Manczinger L, Woo S, Lorito M, Kredics L, Kubicek CP, Druzhinina IS (2007) Genetically closely related but phenotypically divergent Trichoderma species cause green mold disease in oyster mushroom farms worldwide. Appl Environ Microbiol 73:7415–7426CrossRefPubMedGoogle Scholar
  36. Kraus GF, Druzhinina IS, Gams W, Bissett J, Zafari D, Szakacs G, Koptchinski A, Prillinger H, Zare R, Kubicek CP (2004) Trichoderma brevicompactum sp. nov. Mycologia 96:1059–1073CrossRefGoogle Scholar
  37. Kubicek CP, Penttilä ME (1998) Regulation of production of plant polysaccharide degrading enzymes by Trichoderma. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor & Francis, London, pp 49–72Google Scholar
  38. Kubicek CP, Baker S, Gamauf C, Kenerley CM, Druzhinina IS (2008a) Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea. BMC Evol Biol 8:1–16CrossRefGoogle Scholar
  39. Kubicek CP, Komon-Zelazowska M, Druzhinina IS (2008b) Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. J Zhejiang Univ Sci B 9:753–763CrossRefPubMedGoogle Scholar
  40. Kuhls K, Lieckfeldt E, Samuels GJ, Meyer W, Kubicek CP, Börner T (1997) Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences. Mycologia 89:442–460Google Scholar
  41. Kuhls K, Lieckfeldt E, Börner T, Guého E (1999) Molecular reidentification of human pathogenic Trichoderma isolates as Trichoderma longibrachiatum and Trichoderma citrinoviride. Med Mycol 37:25–33PubMedGoogle Scholar
  42. Kullnig CM, Krupica T, Woo SL, Mach RL, Rey M (2001) Confusion abounds over identities of Trichoderma biocontrol isolates. Mycol Res 105:770–771CrossRefGoogle Scholar
  43. Kullnig-Gradinger CM, Szakacs G, Kubicek CP (2002) Phylogeny and evolution of the genus Trichoderma: a multigen approach. Mycol Res 106:757–767CrossRefGoogle Scholar
  44. Lasch P, Nattermann H, Erhard M, Stämmler M, Grunow R, Bannert N, Appel B, Naumann D (2008) MALDI-TOF mass spectrometry compatible inactivation method for highly pathogenic microbial cells and spores. Anal Chem 80:2026–2034CrossRefPubMedGoogle Scholar
  45. Lorito M (1998) Chitinolytic enzymes and their genes. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor & Francis, London, pp 73–100Google Scholar
  46. Lu B, Druzhinina IS, Fallah P, Chaverri P, Gradinger C, Kubicek CP, Samuels GJ (2004) Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum and T. polysporum and their newly discovered relatives. Mycologia 96:310–342CrossRefGoogle Scholar
  47. Muthumeenakshi S, Mills PR, Brown AE, Seaby DA (1994) Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles. Microbiology 140:769–777CrossRefPubMedGoogle Scholar
  48. Neuhof T, Dieckmann R, Druzhinina IS, Kubicek CP, von Döhren H (2007a) Intact-cell MALDI-TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea: can molecular phylogeny of species predict peptaibol structures? Microbiology 153:3417–3437CrossRefPubMedGoogle Scholar
  49. Neuhof T, Dieckmann R, Druzhinina IS, Kubicek CP, Nakari-Setälä T, Penttilä ME, von Döhren H (2007b) Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS. FEBS J 274:841–852CrossRefPubMedGoogle Scholar
  50. Neuhof T, Berg B, Besl H, Schwecke T, Dieckmann R, von Döhren H (2007c) Peptaibol production by Sepedonium strains parasitizing boletales. Chem Biodivers 4:1103–1115CrossRefPubMedGoogle Scholar
  51. Nirenberg HI (1976) Untersuchungen über die morphologische und biologische Differenzierung in der Fusarium-Sektion Liseola. Mitt Biol Bundesanst Land- Forstw 169:1–117Google Scholar
  52. Pignone M, Greth KM, Cooper J, Emerson D, Tang J (2006) Identification of mycobacteria by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. J Clin Microbiol 44:1963–1970CrossRefPubMedGoogle Scholar
  53. Qian J, Cutler JE, Cole RB, Cai Y (2008) MALDI-TOF mass signatures for differentiation of yeast species, strain grouping and monitoring of morphogenesis markers. Anal Bioanal Chem 392:439–449CrossRefPubMedGoogle Scholar
  54. Rifai MA (1969) A revision of the genus Trichoderma. Mycol Pap 116:1–55Google Scholar
  55. Ryzhov V, Hathout Y, Fenselau C (2000) Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Appl Env Microbiol 66:3828–3834CrossRefGoogle Scholar
  56. Samuels GJ (2006) Trichoderma: systematics, the sexual state, and ecology. Phytopathology 69:195–206CrossRefGoogle Scholar
  57. Samuels GJ, Ismaiel A (2009) Trichoderma evansii and T. lieckfeldtiae: two new T. hamatum-like species. Mycologia 101:142–156CrossRefPubMedGoogle Scholar
  58. Samuels GJ, Petrini O, Kuhls K, Lieckfeldt E, Kubicek CP (1998) The Hypocrea schweinitzii complex and Trichoderma sect. Longibrachiatum. Stud Mycol 41:1–53Google Scholar
  59. Samuels GJ, Dodd SJ, Gams W, Castelbury LA, Petrini O (2002) Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus. Mycologia 94:146–170CrossRefGoogle Scholar
  60. Samuels GJ, Dodd SJ, Lu B-S, Petrini O, Schroers HJ, Druzhinina IS (2006) The Trichoderma koningii aggregate species. Stud Mycol 56:67–133CrossRefPubMedGoogle Scholar
  61. Schmidt O, Kallow W (2005) Differentiation of indoor wood decay fungi with MALDI-TOF mass spectrometry. Holzforsch 59:374–377CrossRefGoogle Scholar
  62. Smole SC, King LA, Leopold PE, Arbeit RD (2002) Sample preparation of Gram-positive bacteria for identification by matrix assisted laser desorption/ionization time-of-flight. J Microbiol Meth 48:107–115CrossRefGoogle Scholar
  63. Sperry S, Samuels GJ, Crews P (1998) Vertinoid polyketides from the saltwater culture of the fungus Trichoderma longibrachiatum separated from a Haliclona marine sponge. J Org Chem 63:10011–10014CrossRefGoogle Scholar
  64. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC (2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31:21–32CrossRefPubMedGoogle Scholar
  65. Thrane U, Poulsen SB, Nirenberg HI, Lieckfeldt E (2001) Identification of Trichoderma strains by image analysis of HPLC chromatograms. FEMS Microbiol Lett 203:249–255CrossRefPubMedGoogle Scholar
  66. Tondje PR, Roberts DP, Bon MC, Widmer T, Samuels GJ, Ismaiel A, Begoude AD, Tchana T, Nyemb-Tschomb E, Ndoumbe-Nkeng M, Bateman R, Fontem D, Hebbar KP (2007) Isolation and identification of mycoparasitic isolates of Trichoderma asperellum with potential for suppression of black pod disease of cacao in Cameroon. Biol Control 43:202–212CrossRefGoogle Scholar
  67. Toniolo C, Brückner H (2009) Peptaibiotics – fungal peptides containing alpha-dialkyl alpha-amino acids. Helvetica Chimica Acta, ZurichGoogle Scholar

Copyright information

© German Mycological Society and Springer 2009

Authors and Affiliations

  • Sophie De Respinis
    • 1
  • Guido Vogel
    • 2
  • Cinzia Benagli
    • 1
  • Mauro Tonolla
    • 1
    • 3
  • Orlando Petrini
    • 1
  • Gary J. Samuels
    • 4
  1. 1.Cantonal Institute of MicrobiologyBellinzonaSwitzerland
  2. 2.Mabritec AGBaselSwitzerland
  3. 3.Microbial Ecology Laboratory, Microbiology Unit, Plant Biology DepartmentUniversity of GenevaGenève 4Switzerland
  4. 4.Systematic Mycology and Microbiology LaboratoryUnited States Department of AgricultureBeltsvilleUSA

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